Premature ovarian failure (POF), also know as premature menopause, is defined as the secondary loss of ovarian function before the age of 40 years. POF affects about 1.3 million women in the United States alone. Coulam et al. (1983), Luborsky et al. (1999a). Known contributing factors include irradiation, chemotherapy, and chromosomal abnormalities, although many cases of POF have an unknown origin (idiopathic POF).
Evidence has accumulated that the ovary is a target of an autoimmune process in women with idiopathic POF. Evidence for an autoimmune etiology includes the frequent association of POF with other autoimmune disorders, lymphocytic infiltration of the ovaries, and ovarian autoantibodies in serum. About half of all women with POF express ovarian autoantibodies. Luborsky et al. (1999a).
Ovarian autoantibodies also are detected in sera of women with unexplained infertility. Women with unexplained infertility represent about 15-20% of all infertility cases (about 2% of the population). Forti et al. (1998). Unexplained infertility is defined as the inability to conceive for at least one year, despite normal results on standard tests for reproductive function, which include semen analysis, postcoital testing, ovulation, and tubal patency. Like women with POF, about half of women with unexplained infertility express ovarian autoantibodies. POF may manifest itself initially as unexplained infertility. Farhi et al. (1997).
Autoimmune disorders, in general, are associated with an aberrant immune response against endogenous antigenic determinants, although it is often unclear whether the presence of autoantibodies represents the cause or the effect of cellular pathology. Although the mechanisms underlying ovarian autoimmunity are not completely understood, ovarian autoantibodies presumably are elicited by inappropriate exposure of the immune system to endogenous ovarian proteins. Tung et al. (1995a), Tung (1995b).
The primary diagnostic indicator for an autoimmune disease is the presence of an autoantibody that is directed against a specific endogenous target, or autoantigen, and that is correlated with characteristic clinical features. However, polyclonal activation may occur during the course of the autoimmune reaction, resulting in increased autoantibody titers to a variety of autoantigens. Roitt (1994). For example, in thyroid autoimmunity, there are at least two diagnostically useful autoantigens, peroxidase and thyroglobulin. Thus, while there is a there is a desire to find a specific autoantibody that is a primary diagnostic indicator for POF and unexplained infertility, there is an expectation that multiple autoantigens will be associated with these conditions. Luborsky et al. (1999a).
Diagnosis of Ovarian Autoimmunity, Idiopathic POF, and Unexplained Infertility
Ovarian autoimmunity may be present 8-10 years before the manifestation of clinical symptoms. Thus, premature menopause and unexplained infertility may represent the endpoint of a slow, degenerative process with subclinical intermediate symptoms. Farhi et al. (1997). Accordingly, early diagnosis of autoimmune disease allows treatment to maintain or restore ovarian function before a clinical manifestation of infertility or POF.
Unfortunately, diagnosis of ovarian autoimmunity has presented significant problems. Current methods of assessing ovarian function rely on evaluation of the pattern and length of menstrual cycles, and measurement of estradiol and follicle stimulating hormone (FSH) levels in serum during the early follicular phase. Menstrual cycle evaluation is subjective and is generally indicative only of the final clinical manifestations of ovarian failure. Measurement of serum endocrine levels does not differentiate between endocrine or autoimmune etiologies for ovarian dysfunction. Luborsky et al. (1999b), Luborsky et al. (1998).
Recently, it has been possible to diagnose ovarian autoimmunity by detecting the presence of ovarian autoantibodies. The presence of ovarian autoantibodies is correlated with poor estradiol production in response to hormone therapy and with poor pregnancy outcome following in vitro fertilization (IVF). Luborsky et al. (1999a), Meyer et al. (1990), Gobert et al. (1992), Barbarino-Monnier et al. (1991). In contrast, presence of ovarian autoantibodies does not correlate with FSH levels, indicating that detection of ovarian autoantibodies provides an assessment of ovarian failure independent of routine endocrine assessment. Luborsky et al. (1999b), Luborsky et al. (1998). The presence of ovarian autoantibodies is highly correlated with POF and unexplained infertility, but not other categories of infertility. McKenna et al. (1999), Luborsky et al. (1999c). Thus, detection of ovarian autoantibodies provides a reliable indicator of an ongoing ovarian disease.
Methods of Detecting Ovarian Autoantibodies
Previously, the only method of detecting ovarian autoantibodies was immunohistochemistry, which is subjective, labor intensive, and qualitative. Immunohistochemistry necessitates the use of thin sections of ovarian tissue. Since there are rarely more than a few active follicles expressing antigens of possible relevance to fertility in the whole ovary, the variability from section to section is extremely high.
Recently, however, it has been possible to screen sera for ovarian autoantibodies using an enzyme-linked immunosorbent assay (ELISA). Luborsky et al. (1990), MacCorkle et al. (1999). In this assay, human sera are contacted with immobilized ovarian antigens. Autoantibodies against ovarian antigens are detected by the formation of immune complexes with the immobilized antigens. The ease and feasibility of using this assay is increased by the recognition that rat ovarian tissue can be used as a source for antigens to identify ovarian autoantibodies (correlation greater than 95%). Thus, at least one major antigenic determinant in ovarian tissue is structurally conserved between rat and human antibodies, such that the human ovarian autoantibodies are capable of reacting with rat antigen(s). This ELISA can be used in conjunction with standard reproductive endocrine level tests to diagnose or predict POF or unexplained infertility. The ELISA further can be used to measure the progress of treatment of POF or unexplained infertility, by such methods as immunosuppression with glucocorticosteroids. Luborsky et al. (1990), Hoek et al. (1997). As one alternative to immunosuppressive therapy, individuals positive for ovarian autoantibodies may choose to bear children sooner, before it becomes difficult or impossible due to loss of ovarian function.
Identification of an Antigen Associated with an Ovarian Autoimmune Etiology for Infertility
Little is known of the identity of the ovarian determinants that interact with human autoantibodies. One line of evidence suggests that ovarian antigens may be diverse. Immunohistochemical staining of rat ovarian tissue sections indicates antigenic determinants in the nucleus, cell membrane, and cytoplasmic organelles, suggesting multiple antibody specificities in autoimmune sera. Meyer et al. (1990).
The identification of a determinant that is unique to ovarian autoimmunity would have significant predictive and diagnostic value. Once identified, an antigen could be isolated, thus providing an inexpensive and standardized source of an antigen for diagnostic screens. A diagnostic procedure, based on detection of such an antigen, could identify individuals at risk of POF or unexplained infertility before the onset of ovarian dysfunction. Alternately, such a diagnostic procedure could permit evaluation of the progress of therapy to treat or reverse ovarian dysfunction.
Two endocrine glands synthesize steroid hormones, the ovary and the adrenal. Steroid-producing cells (SPCs) produce steroids in both of these glands. SPCs in the two glands utilize partially overlapping sets of metabolic enzymes to produce different steroid hormones. Both glands express the P450 side chain cleavage enzyme (P450 scc) in an initial step in steroid hormone biosynthesis. However, 21-hydroxylase (21-OH) is expressed abundantly in the adrenal but not in the ovary. By contrast, 17-alpha hydroxylase (17-OH), in humans, is expressed at high levels in the ovary but not in the adrenal. Dufau et al. (1997).
Autoantibodies against the SPC antigens 17-OH, P450 scc, and 21-OH have been found in sera of patients with POF associated with autoimmune adrenal disease (polyglandular disease), but not in sera of POF patients without polyglandular disease. Chen et al. (1996). Autoantibodies that recognize these SPC antigens were detected by the formation of immune complexes with radiolabeled enzymes that were prepared by in vitro transcription. Autoantibodies against these SPC antigens were detected in patients with type I or type II polyglandular syndrome (APS), adrenal cortex antibody (ACA), and Addison""s disease (adrenal insufficiency). Significantly, no antibodies to 17-OH, P450 scc, or 21-OH were found in POF patients without adrenal autoimmunity, except for one serum containing low levels of anti-17-OH antibodies. Thus, it would appear from this data that these autoantibodies are associated with an adrenal etiology in polyglandular syndrome but not with an ovarian etiology in POF and unexplained infertility.
Thus, there is an ongoing need to identify autoantibodies associated with an ovarian etiology in POF and unexplained infertility. This is especially important because Addison""s disease is relatively rare, and POF associated with adrenal autoimmunity accounts for only about 2-10% all cases of idiopathic POF. Hoek et al. (1997).
Only limited progress has been made in finding an antigen specifically associated with idiopathic POF that occurs in the absence of polyglandular diseases. Potentially, one such antigen is 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), another enzyme involved in steroid metabolism. Arif et al. (1996). Although autoantibodies against 3 beta HSD apparently are associated with idiopathic POF occurring in the absence of polyglandular disease, these autoantibodies appear in only 10 of 48 (21%) POF sera. Not only does 3 beta HSD appear with a low frequency in POF sera, but this enzyme is also present in adrenal tissue. Thus, the presence of 3 beta HSD autoantibodies is a relatively poor indicator of an ovarian etiology in autoimmune infertility.
Thus, there is a current need in the art to define a specific antigen that is diagnostic for a majority of POF patients with ovarian autoimmunity that occurs in the absence of polyglandular disease.
Autoantibodies to a CYP17 protein (17-alpha-hydroxylase, or 17-OH) are found in a majority of sera from patients with unexplained infertility and POF, as described herein. It has been discovered that anti-CYP17 autoantibodies are indicative of an ovarian etiology of POF that is unassociated with polyglandular disease. This strong correlation between anti-CYP17 autoantibodies and ovarian autoimmunity allows a quick and efficient diagnosis of ovarian autoimmunity by detecting the presence of this autoantibody in a sample from a test subject.
Accordingly, the invention provides a method of diagnosing an ovarian autoimmune etiology of unexplained infertility or POF, comprising contacting a biological sample, preferably serum, from a patient and detecting an anti-CYP17 autoantibody, preferably through detecting the formation of an immune complex between the autoantibody and CYP17. In one embodiment, the method is applied to women manifesting POF or unexplained infertility, or suspected of manifesting either, in the absence of polyglandular disease.
The formation of an immune complex may be detected in any of a number of ways. In one embodiment, a detectably labeled CYP17 protein is provided. Alternately, the immune complex may be detected by forming a second immune complex between the anti-CYP17 autoantibody and a detectably labeled secondary antibody that binds immunoglobulin, preferably the immunoglobulin backbone of the autoantibody. In another embodiment, the autoantibody is detected by formation of an immune complex with a detectably labeled anti-idiotype antibody that specifically recognizes the complementarity determining region (CYP17-binding region) of the autoantibody.
The invention further provides a method of diagnosing idiopathic POF or unexplained infertility in an individual, comprising:
(a) contacting the CYP17 protein immobilized on a support with a test sample comprising an anti-CYP17 autoantibody, and
(b) determining whether an immune complex forms between the CYP17 protein and the anti-CYP17 autoantibody,
wherein the determining is accomplished by contacting the immune complex with a detectably labeled anti-immunoglobulin antibody that recognizes the anti-CYP17 autoantibody. Alternately, this diagnostic method may comprise:
(a) contacting a tissue extract comprising a CYP17 protein with an antibody that is capable of binding the CYP17 protein, then
(b) removing immune complexes formed between the CYP17 protein and the antibody that is capable of binding the CYP17 protein, then
(c) immobilizing the tissue extract on a solid support,
(d) contacting the immobilized tissue extract with a test sample comprising an anti-CYP17 autoantibody,
(e) determining whether an immune complex forms between the CYP17 protein and the anti-CYP17 autoantibody, and
(f) comparing the amount of immune complex with a control tissue extract that was not contacted with an antibody that is capable of binding a CYP17 protein,
wherein the determining is accomplished by contacting the immune complex with a detectably labeled anti-immunoglobulin antibody that recognizes the anti-CYP17 autoantibody.
The reagents may be packaged in the form of a kit containing instructions for using the reagents to diagnose ovarian autoimmunity in an individual having, or suspected of having, idiopathic premature ovarian failure POF or unexplained infertility. These reagents will include a reagent that specifically recognizes an anti-CYP17 CYP17 autoantibody. Such a regeant may include a detectably labeled CYP17 protein or a detectably labeled anti-idiotypic antibody that specifically recognizes the complementarity determining region (CYP17-binding region) of the autoantibody. Preferably, one or more of the reagents are immobilized on a solid support.